Hepatitis C Virus (HCV) is now recognized as being the primary cause of transfusion-associated non A, non B (NANB) hepatitis. HCV is a single stranded, positive sense RNA virus with similarities to flaviviruses and pestiviruses (Miller R. H. and Purcell R. H. Proc. Natl. Acad. Sci. 87: 2057 (1991); Weiner A. J., et al., Virology 180: 842 (1990)) and is in global distribution. Although the acute presentation of HCV is generally mild, with only 25% of patients developing jaundice, a large proportion (>50%) of infected individuals go on to develop chronic hepatitis with serious and potentially life threatening sequelae such as cirrhosis and hepatocellular carcinoma (Jove J., et al., Liver, 8: 42 (1990); Hopf U., et al., Hepatology, 10: 69 (1990)).
Infection with HCV is currently diagnosed by direct detection of viral RNA by PCR or by detection of anti-HCV antibodies (generally to the HCV structural core protein or non-structural NS3 protein). In general it can take up to about 70 days after individual has been infected with HCV before the individual develops antibodies to the virus, therefore, antibody tests alone during this 70-day period cannot determine whether HCV infection has occurred. Viral RNA, on the other hand, can be detected about 10 days following infection, i.e. the “window period” for nucleic acid testing (NAT) is much shorter than for antibody testing. However, NAT tends to be less cost-effective than antibody testing and is prone to handling errors such as contamination. In addition, RNA levels can drop below the limit of detection for this nucleic acid testing when the initial peak of virus resolves, especially when testing pooled samples.
U.S. Pat. Nos. 6,596,476; 6,592,871; 6,183,949; 6,235,284; 6,780,967; 5,981,286; 5,910,404; 6,613,530; 6,709,828; 6,667,387; 6,007,982; 6,165,730; 6,649,735 and 6,576,417 describe antigens based on core protein and their use to detect HCV.
More recently, HCV antigen assays have been developed which demonstrate that HCV core protein antigens can be detected in a sample significantly sooner than antibodies can be detected. Studies have shown that the average time from the first viremic bleed to the first HCV antigen positive bleed is estimated at 2.0 days and that the average time to the first HCV antibody positive bleed at 50.8 days (Couroucé A. M., et al., Transfusion, 40, 1198-1202 (2000)).
A combination assay that detects HCV antigens and anti-HCV antibodies would, therefore, provide a means of detecting HCV infection within the above-noted 70-day window period, and would also identify exposure to HCV after seroconversion. An earlier diagnosis of HCV could help to prevent transmission of the virus from an infected individual to others individuals, and to minimize the risk of contamination of the blood supply. In order to develop a suitable combination assay, however, the problem of interference or cross-reactivity with respect to assaying an antigenic protein and antibodies to the same antigenic protein in a single assay must be addressed. Interference or cross-reactivity results when, for example, an HCV antigenic protein bound to a solid phase is used to capture anti-HCV antibodies in a sample. Because the HCV antigenic protein has the same epitopes as those recognized by the labeled antibody or antibodies used to detect the HCV antigenic protein in the sample, the labeled antibody or antibodies can also bind to the HCV antigenic protein on the solid phase thus giving a false positive response even in the absence of test sample.
Various combination assays to detect HCV infection have been described which use different strategies to overcome the problem of interference. International Patent Application No. PCT/US02/19958 (WO 03/002749) and U.S. Pat. Nos. 6,727,092 and 6,855,809 each describe a combination assay to detect HCV which uses monoclonal antibodies to detect core proteins from a sample, and core peptides or recombinant core proteins to capture antibodies to core proteins from a sample. The recombinant core proteins have been engineered such that the epitopes recognized by the monoclonal antibodies are eliminated or modified. In one example, the described combination assay uses a single peptide of 17 amino acid residues in length, corresponding to amino acids 11 to 28 of core protein to capture antibodies to core proteins from a sample. The preferred format, however, uses recombinant core proteins corresponding to amino acids 1 to 100 or 8 to 100 of the core protein sequence that have been engineered to delete the epitopes recognized by the monoclonal antibodies used in the assay.
International Patent Application No. PCT/FR03/01429 (WO 03/095968) and U.S. patent application Ser. No. 10/431,587 (US2004/0072267) describe another combination assay to detect HCV in which certain epitopes of the target antigens used to capture the antibodies are structurally modified and destroyed. The antibodies used in the assay are then specifically selected such that they precisely recognize the corresponding unmodified epitopes and thus cannot bind to the modified antigens, which no longer exhibit these same epitopes. A combination assay kit (MonoLisa® HCV antigen-antibody Ultra, Bio-Rad Laboratories) based on this approach has been developed and has been determined to be an improvement over antibody-only based assays, but is still less sensitive than NAT (Ansaldi, F., et al., J. Viral Hepatitis, 13:5-10 (2006); Laperche, S., et al., Transfusion, 45:1965-1972 (2005); Laperche, S., et al., J. Clin. Microbiol., 43:3877-3883 (2005)).
Similarly, US 2003/0108563 and US 2003/0152965 describe another combination assay in which the HCV core protein employed as antigen comprises a sequence in which amino acid residues other than the HCV core protein residues 10-43 are altered or deleted, and anti-HCV core antibodies are employed that do not recognize the modified HCV core protein.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.
All patents and publications referred to herein are hereby incorporated in their entirety by reference.